The long-term goal of the proposal is to understand the fundamental mechanisms through which GATA factors control development of the heart. Recently we have produced mouse embryos from embryonic stem (ES) cells in which both Gata4 and Gata6 genes, which encode zinc finger transcription factors, were simultaneously disrupted. Examination of these embryos revealed that cardiac myocyte differentiation was completely blocked and the resulting embryos exhibited acardia. Although these data are provocative we do not know i) whether this requirement for either GATA4 or GATA6 during the onset of heart cell differentiation is conserved in humans, ii) whether redundant roles for GATA4 and GATA6 also exist for maintaining cardiac myocyte gene expression in terminally differentiated cardiac myocytes, or iii) if the dependency of cardiac myocyte differentiation on both GATA4 and GATA6 reflects only a cell autonomous role. These questions will be addressed in three specific aims: Aim 1: Do GATA4 and GATA6 have evolutionary conserved roles in controlling cardiac myocyte differentiation? The evolutionary conservation of the roles of GATA4 and GATA6 during development will be examined using human ESCs as a model of cardiomyocyte specification and differentiation. Aim 2: Do GATA4 and GATA6 have functionally redundant roles in controlling a transcription factor network that is essential for maintaining cardiac myocyte gene expression? This will be tested using a combination of gene array and chromatin immunoprecipitation analyses on primary cardiomyocytes in which GATA4, GATA6, or both GATA4 and GATA6 have been ablated. Aim 3: Do GATA 4 and GATA6 act in multiple tissues to ensure the completion of cardiogenesis? The role of GATA4 and GATA6 in the cardiac myocytes and endoderm will be evaluated using conditional knockout mice and human ES cells.